Apparatus for and a process of treating immunized sera



E. CARDONE July 6, 1937 APPARATUS FOR AND A PROCESS OF TREATING IMMUNIZED SERA Filed Nov. 14, 1934 ATTORNEY.

Patented July 6, 1937' PATENT OFFICE APPARATUS FOR. AND A ritocEss or TREATING IMMUNIZED SERA Eugene Oardone, Brooklyn, N. Y. Application November 14; 1934, Serial No. 752,994

5 Claims.

My invention relates to apparatus for and aprocess of treating immunized sera. More particularly, it relates to apparatus for and a process of refining and concentratingsera containing antitoxin or antibacterial material and reducing the inert protein content therein to render the sera stable and more powerful. In the methods now used for treating sera, considerable difllculty has been experienced in removing the major portion of inert proteins in orderto obtain suitable concentrated form of serum. II a large quantity of such proteins were removed by presently known methods the cost or the treated serum would be excessive. At the same time, there isa highlossof anti-toxic or antibacterial content. A serum treated 'by known-methods and/or apparatus, eventually may become cloudy due to precipitation of inert bodies. 01d .serum cannot be easily reconcen-j trated by present methods; If such sera were reconcentrated, there would be considerable losses in 'antitoxin or antibacterial body content because all the inertproteins have been previously removed. V

It is one of the objects of invention to provide easily operable apparatus for transforming some or all oi the proteins in a serum.

It is one other object of the invention herein, to provide an economhz process whereby a com,- paratively clear serunrot comparatively low per-- centage of nitrogen er unit of antitoxin or antibacterial bodies,- is b tained.

It is another objec t \ot the invention to provide a process for transforming the water soluble proteins in an immunid serum to water insoluble proteins and eliminating suitable quantities ofsuch proteins-Ironnthe serum without apprecie able losses of antitoxin or antibacterial bodies.

One further a invention is to pH concentrationfito remove at will any undesirable proteins orgfactions therefrom, at differentpH concentr 'ons without adversely aiIecting the antitoxi or antibacterial body content and to render sue sera stable andclear.

One other object of the -invention is to concentrate d sera, whether previously concentr or not, without the use-ofprecipitating saltab t by action of electrical pH variants or byj addition of an alkaline substance. Another obj t oi! the invention is to providean immunized serum'having-a low'proteincomtent of antitoxin or antibacterial body more specific object of this ect sera containing antitoxin or antibacterial males to; the action or various whether ipreviously concentrated or .not, to a serum or body extracts,-

transforming action by means of novel apparatus herein disclosed, in which the pH value .0! the.

. serum is varied and water soluble proteins are.

converted to water insoluble proteins and .pree cipitated; removing said undesirable insoluble proteins or any -fractions thereof, then treating the serum with alkali to change the pH value of the same and precipitating the proteins from the serum, which, contain the antitoxin or antibacterial bodies. The process further includes treating of the precipitate formed with acid substances down to a desired pH value to dissolve said precipitate. and thenadding solid salt and raising the pH value of theresultant "solution with alkaline substances to stabilize the same preparatory to subsequent treatment for sterilization and standardization.

In the-dra'wingr q Fig. 1 is a diagrammatic view of the novel ap- Fig; 2 is a modification of a part 01'- theapparatus shown in Fig. -1. v

In the drawing similar reference characters designate like parts throughout.

An immunized serum containing antitoxins and which is to be treated may have been previously concentrated, for example, by using ammoniunrsulphate' or some other salts. In such case, I dialyze-the serum to remove ,crystalloids therefrompand add suficient distilled water to dilute thereby reducing the protein per cubic centimeter of serum. In cases where the serum is obtained for treatment directly from live stock, particularly horses, such previous concentration is not necessary.

\At this point, it is desirable to. describe the 1 In the following steps, the serum is now treatnovel apparatus comprising part of-the invention and shown in the drawing before the next step of known manner.

the process. A pair of containers II and' I2, consisting of porous or semi-permeable membranous material, each filled with water, are mounted on to metal tubes I3 and II respectively by any suitable members, as shown in the drawing. Said metal tubes are: filled with water and accommodate platinum electrodes I 5 and I 6 respectively, and may be supported in any well Each electrode extends outwardly through stoppers l1 and I8, respectively, and is insulated at said upper extended portion. The stoppers may be provided with a plurality of openings to permit escape of gases generated in the tubes during their use.

One of the electrodes is in'connection with wiring of a source of electric current, while the other electrode is in connection with a variable resistance' coil 19. The latter is in connection with a fixed resistance 20. A series of carbon lamps 2| are in connection with the coils in order to help regulate the electric current to any desired ainperage.

During the passage of the current from one electrode to the other, gases are generated in containers ll and I2 and in tubes l3 and M. The gases generated in said'containers pass thru the pores thereof or thru a slit previously made at the top of the containers prior to their mounting on the tubes as shown in the drawing.-

After having placed the serum into container l0 and the bags filled with water set up on the tubes l3 and M, as shown in the drawing, the current is turned on to any desired amperage regulated by the variable resistance coil IS.

The electric current passing through the electrodes, water and serum in the manner according to the above described apparatus, transforms all the'water soluble proteins originally present in the serum, into water insoluble proteins up to a certain point. I have found that by passing current through the serumin the manner above described, the pH value of the serum rises and precipitation of .the water soluble proteins sets in. I stop this transformation when theserum reaches a pH value of about 5.9-6.1, which is about the most desirable ionic concentration range at which to stop. It is to be noted that thistransformation does not adversely affect the antitoxin material. The serum at this pointhas a definite precipitate formed as a result of the transformation. Once the transformation is complete, as shown by the: above pH range, in order to remove a large percentage of water imsoluble proteins'or transformed glqbulins, the precipitate-is-separated by centrifuging or filtering. The filtrate contains the antitoxin material with the transformed proteins, while the residue contains the water insoluble proteins-and some of the adhering antitoxin material. The precipitate is washed with distilled water in order to take up theadhering antitoxin material. The washings are added to the original filtrate while the washed precipitate is discarded.

The total filtrates above, are then treated with alkaline solution until a pH value of about 6.7-7.0 is obtained. This bringing up of the pH value causes the precipitation of proteins which remained in solution after the transformation step above, and also of antitoxin material.

By my process, it is also possible to add alkali at-any. intermediate pH value. after pH 5.9 up' to 7.0 in order to take ,down any desired fractions of proteins, instead of only transforming by the electrical means above described.

' The precipitated proteins and antitolrin mate-- rial are filtered off from the alkaline mixture and expressed to remove any traces of the filtrate which is discarded. The expressed precipitate is dissolved with acetic acid until pH values between 5.4-5.2 are obtained. To the resultant solution is added sufficient solid sodium chloride until there is a concentration of this salt of about 1%. The pH value of the solution is brought up to about 6.8 by adding alkaline solution in order to stabilize and preserve the same.

The final product is clearand ready for further treatment, namely, sterilization and standardization.

In the treatment of antibacterial sera the steps are the same as those in the case of serum containing antitoxin material, except that after dialysis, upon the addition with acetic acid, the pH value is brought to a range between 4.2-5.1, instead of 5.1-5.4, as in sera with antitoxin material. The serum is then treated electrically the same as with respect to the serum containing antitoxin material, until a suitable pH value of 5.9 is reached to eifect transformation. There is a precipitate of some of the transformed proteins. I then filt'er to separate the precipitate. Then I acidify the precipitate with any acid such as acetic acid to dissolve the antibacterial material;

while the inert proteins remain in the form of a precipitate. The dissolved antibacterial material is filtered oif and added to the-original filtrate obtained as a result of the transformation up to pH 5.9-6.1. may be washed with distilled water to recover any traces of adhering antibacterial bodies. These washings are added to the first or original fil- The precipitate furthermore to cause a fall in pH value from 7.0 to pH 2.0-3.0

to precipitate'some inert proteins.

From-the foregoing description taken in connection with the accompanying drawing, it will be noted ,that I have devised apparatus and a process embodyi g my invention for refining antiserum, thereby reducing the total inert solids content therein. Also, by'my invention I obtain a serum which Iias'a low protein content per unit of antitoxin or antibacterial body content. ,I can also control the amount of precipitation of proteins by regulating the electroionio concentration of the serum.

Where other methods fail to effect reconcentration without substantial losses, by my method I can economically increase the. total potencyover 25% without any appreciable loss in antitoxin or antibacterial body content.

, WhileI have 'described a preferred form of my invention, it is to be understood that various modifications as to steps, parts, choice of materials and their arrangement may be made without departing from the spirit and scope of the invention as defined in the appended claims.

.I claim: V I

1. Apparatus for transforming water soluble proteins into water insoluble proteins in antisera, comprising a plurality of metal tubes containing an electrical conducting liquid medium, porous containers for said tubes for containing water, an electrode insertible into each of said metal tubes, a container with serum for said porous 75 containers and variable electrical control means in connection with said terminals ,for permitting the passage of electrical current through the antisera.

2. Apparatus for transforming water soluble proteins into water insoluble proteins in antisera, comprising a pair of metal tubes filled with water, an electrode extending into each of said tubes and concentric therewith, means carrying each electrode and insertible into each tube, a porous container filled with. water for each of said tubes and mounted thereon, a container filled with serum accommodating said porous containers, and an electric circuit of variable resistance in connection with the terminals for regulating the amperage passing through the ter-' minals.

3. In apparatus for variably transforming water soluble proteins into water insoluble proteins tric conducting terminals e in antisera, the combination with a pair of elecmetal tube filled with water, of a porous water filled container on each of said tubes, a containquit.

extending into a 4. In apparatus for treating immunized sera, the combination with a container having serum, of a pair of platinum electrodes/:1 metal tube containing water for retaining each of said electrodes, a porous water filled container accomtion with the other platinum electrode.

5. Apparatus for treating immunized sera, comprising a container for serum. a plurality of semi-permeable container members filled with water and immersed in said serum, a metal tube EUGENE 25 

